This invention relates to a composition which is either polystyrene or a substituted polystyrene crosslinked with divinylbenzene and swollen by N-methyl-2-pyrrolidone.
Briefly, peptides are polyamides comprising two or more amino acids. Synthetic peptides generally are prepared by well-established procedures which require the blocking of various reactive groups in order to minimize degradation and side reactions. The blocking groups then are removed as a last step in the synthetic procedure, with purification procedures normally being carried out prior to deblocking.
Peptide synthesis usually involves fragment condensation methods which can be summarized as follows. A plurality of amino acid residues are condensed via peptide linkages to give a fragment, A-COOH, terminating in a carboxylic acid group and wherein all other reactive groups are blocked. Similarly, an amine-terminated fragment, H.sub.2 N--B, is prepared. The two fragments then are combined to give A--CONH--B, which in turn can be used as an intermediate to prepare a higher molecular weight peptide or deblocked and used per se.
Blocked peptides consisting of more than about four amino acids usually are insoluble in most solvents, water included. Such blocked peptides are, however, soluble in the high-solvency liquids which include N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMAC), dimethylsulfoxide (DMSO), hexamethylphosphoric triamide (HMPA), and the like. But blocked peptides are difficult to purify by crystallization because the blocked peptide fragments, A--COOH and H.sub.2 N--B, and the final product prepared therefrom, A--CONH--B, have similar solubilities, a problem not eliminated by deblocking.
Fortunately, blocked peptides can be purified by chromatographic techniques. Ideally, such techniques will be passive, as contrasted with, for example, ion exchange chromatography. However, typical prior art passive procedures, such as adsorption chromatography utilizing DMF as solvent and a hydrophilic, insoluble, crosslinked dextran as the resin, generally are satisfactory only for blocked peptides having fewer than about eight amino acid residues.
Theoretically, gel exclusion chromatography (synonymous with gel filtration and gel permeation chromatography) is an ideal system for the separation of blocked peptides. However, most solvents suitable for swelling the resin, which solvents include, among others, benzene, toluene, and tetrahydrofuran (THF), are poor solvents for blocked peptides.
It will be recognized by those skilled in the art that gel exclusion chromatography requires that the resin be swollen by the solvent, for it is this swelling which produces the voids by means of which the separation of blocked peptides occurs. Furthermore, increasing the degree of swelling generally increases resolution, a term employed herein to mean separation of the final blocked peptide from the blocked peptide fragments or intermediates. Hence, the problem heretofore has been the lack of a good blocked peptide solvent which also is capable of adequately swelling a suitable resin, which resin usually is polystyrene crosslinked with divinylbenzene. In the typical prior art procedures mentioned hereinbefore, there is only minimal swelling of the resin by the solvent.
Resin swelling also is important in the recently developed procedure known as solid-phase peptide synthesis. Solid-phase peptide synthesis is based upon the premise that amino acids can be assembled into a peptide chain of any desired sequence while one end of such chain is anchored to an insoluble support. Most commonly, the insoluble support is a substituted polystyrene crosslinked with divinylbenzene, wherein the polystyrene is substituted with an appropriate anchoring group, i.e., a group capable of reacting with the amino or carboxy terminus of an amino acid or peptide to give a covalent bond.
The potentially reactive anchoring group remains unreactive when in a nonsolvated state. The role of swelling of the insoluble support becomes more fully appreciated upon recognition of the general resemblance between swollen and dissolved states. Indeed, swelling is a process of incipient dissolution of segments between crosslink sites within the polymer network. Thus, the success of solid-phase procedures is to a large extent dependent upon the appropriate solvation of the anchoring group loci.
Under ideal solvation or swelling conditions, both the nonpolar, substituted polystyrene support and the more polar peptide chains are in an essentially extended configuration, thereby permitting ready access by reagents. In the nonpolar solvents commonly employed, such as methylene chloride, the substituted polystyrene is extended, while the peptide chains are not. The reverse situation occurs in the presence of polar solvents, such as acetic acid, DMF, alcohols, and the like. Thus, ideal swelling conditions have not heretofore been attainable.